When cells are extracted from mammalian tissues and placed in culture, they tend to behave as either anchorage dependent or anchorage independent. Anchorage dependent mammalian cells grow by first attaching to a surface. These cells often divide until the surface on which they are attached is fully covered i.e. they divide and aggregate to form a confluent monolayer. Examples of anchorage dependent cells that are commonly used in industrial and research settings include Chinese Hamster Ovary cells (CHO cells) and L-929 Murine Lung Fibroblast Cells. Anchorage independent cells, however, do not require a surface on which to attach prior to dividing. Rather, after being placed in culture, they grow in suspension either as single cells, or as clusters of cells which are often referred as suspended spheres or suspended aggregates of cells. Examples of this include neural stem cells and mammary stem cells.
A substantial proportion of the volume within a suspended aggregate in culture is comprised of space between the cells (similar to tissues). Most of this extracellular space is filled with an intricate network of proteins and polysaccharides collectively termed an extracellular matrix (ECM). The molecules which make up the matrix can be divided into two categories: extracellular matrix molecules (ECM molecules) and cell adhesion molecules (CAMs). ECM molecules (comprised of collagen, proteoglycans and non-collagen glycoproteins) are secreted into the extracellular space where they are assembled into a complex mesh that remains closely associated with the cell that produced them. Since they are produced locally, the amount and composition of the ECM can vary considerably throughout an aggregate or a tissue. Due to the massive quantities of ECM in connective tissues such as bone, tendon, cartilage, and dermis, it was originally believed that the function of the ECM was simply to provide inert physical support. However, recent evidence shows that the ECM is a complex and dynamic entity that can regulate the survival, development, migration, proliferation, shape and function of the cells in contact with it (Alberts et al., 2002).
In addition to ECM molecules, CAMs are also known to exist between both anchorage dependent cells and anchorage independent cells. The term ‘cell adhesion’ implies that these molecules are a form of intercellular glue. Whereas they do bind cells in close proximity to one another, they have very important roles in tissue development and inter- and intracellular signaling. CAMs can be subdivided into four different families based on structural homology. They are the immunoglobulin superfamily of CAMs (IgCAMs), cadherins, selecting, and integrins.
CAMs can interact with one another, and with ECM molecules. Many ECM molecules and CAMs have important secondary and tertiary structures, and their conformation in the extracellular matrix allows them to interact through the formation of weak noncovalent bonds such as hydrogen bonds, and stronger bonds such as ionic bonds mediated by divalent cations, or covalent disulfide bridges between amino acid residues. These intercellular interactions cause cells in a confluent monolayer or a suspended aggregate to bind very strongly to one another.
Most tissues such as the pancreas and the brain are made up of billions of cells that are held together by ECM molecules and CAMs. In order to isolate cells from these primary tissues for cell culture, it is necessary to dissociate the tissues into a single cell suspension prior to being placed into culture. Once in culture, most cells tend to divide and/or reassociate with each other to form monolayers or suspended clusters. Even hematopoietic cells (which are typically present as a single cell suspension in vivo) tend to aggregate when manipulated in culture. If it is desired to maintain actively proliferating cells in culture beyond a few days, it is necessary to subculture the cells (i.e. remove them from one culture vessel, and place them at a lower cell concentration into a new culture vessel containing fresh medium). In order to do this in anchorage dependent cultures, monolayers of cells have to first be detached from the surface on which they are attached, and then the detached monolayers have to be dissociated into a single cell suspension. In anchorage independent cultures, the suspended clusters of cells have to be dissociated into a single cell suspension. The single cell suspension can then be used to inoculate a new culture vessel.
In addition to isolating cells from primary tissues, and subculturing cells in existing cultures, the generation of single cell suspensions is extremely important for a variety of applications. For example, during cell therapy, single cells are delivered to certain sites in order to treat specific conditions. Transplanting aggregates is undesirable because (i) aggregates can plug the delivery device (ii) it is difficult to estimate the number of actual cells that are delivered (iii) cells in aggregates are more susceptible to cell death due to the nutrient and oxygen mass transfer limitations that they suffer and (iv) aggregates are less likely to migrate to areas of damage, respond to local cues, and integrate into the host cellular architecture. Single cells are also necessary for basic biological research. For example, cell sorting methods are used to determine the composition of heterogeneous cell populations, and to isolate specific subpopulations of cells with desirable characteristics which can then be used to conduct further research, or used therapeutically in a clinical setting. Cell sorting methods can only be used effectively on single cells. The generation of a single cell suspension also has applications in other areas such as the production of bio-molecules and clinical diagnostics.
Several methods have been developed to generate single cell suspensions from primary tissues, attached cells in culture, and aggregates in culture reviewed in (Freshney, 2000). These methods involve the use of physical forces (mechanical dissociation), enzymes (enzymatic dissociation), or a combination of both. Mechanical means of detaching cells that are attached to a surface include the use of cell scrapers. Mechanical means of separating cells which are attached to one another include trituration through a narrow bore pipette (Reynolds and Weiss, 1992; Sen et al., 2001), fine needle aspiration (Ottesen et al., 1996), vortex disaggregation (Vos et al., 2003), and forced filtration through a fine nylon or stainless steel mesh. Whereas all of these methods are effective in creating single cell suspensions, the excessive physical forces involved often result in a significant amount of cell death and cell damage. In situations where the generation of a suspension of viable single cells is the ultimate goal, cell death and cell damage are extremely undesirable.
Mechanical dissociation can also result in the death of specific groups of important cells within a heterogeneous population. For example, larger cells are known to be more sensitive to shear than smaller cells. Continually killing specific cell types during serial passaging could be detrimental to a cell line during long term culture. In addition, the death of specific cell types could adversely impact results derived from procedures that rely on the generation of a single cell suspension such as flow activated cell sorting, and clonal and population analyses in the promising area of stem cell biology. Moreover the manual nature of certain mechanical dissociation protocols (e.g. trituration, which is done by hand) often make it difficult compare measured values (such as cell viability) from different sources since dissociation efficiency varies between individuals. In fact, the manual nature of this procedure may contribute to differences in the physical attributes (e.g. cell concentration, cell viability, cell size distribution etc.) between two otherwise identical samples.
In an attempt to avoid the negative consequences of mechanical dissociation, researchers have used enzymes (either alone or in combination) which are directed towards one or more components in the ECM. Certain enzymes are known to target and cleave specific molecules present within the ECM. For example, the enzyme trypsin (which cleaves polypeptide chains on the carboxyl side of arginine and lysine residues) is commonly used to detach and dissociate monolayer cultures, whereas collagenase is often used to dissociate primary tissues and aggregates. However, not all cell types can be easily dissociated using enzymes. For those cell types that are susceptible to enzymatic dissociation, it has been shown that enzymes can be detrimental to the cells and negatively impact the ability of the generated single cells to subsequently survive and/or divide. For example, when neural stem cell (NSC) aggregates were dissociated using trypsin, the growth rate of the single cells in subsequent culture was found to have been adversely affected relative to single cells generated using mechanical dissociation (Sen, 2003). This result may be attributable to the fact that trypsin is known to cleave certain classes of cell surface transmitter receptors (Allen et al., 1988). In the extreme, enzymes can completely destroy cells. For example, collagenase has been shown to reduce viable cells to debris when used to dissociate neural stem cell aggregates (Kallos et al., 1999).
Human embryonic neural stem cells inoculated into serum free medium can be induced to divide and form aggregates over time. Visually, the aggregates contain a significantly greater amount of extracellular matrix compared to embryonic neural stem cell aggregates derived from mice. Currently, the state-of-the-art method of generating a single cell suspension from these aggregates involves mechanical dissociation. However, due to the large quantities of extracellular matrix, mechanical dissociation of human neurosphere aggregates results in a much greater cell death relative to that caused during the mechanical dissociation of murine neural stem cell aggregates. Even in the hands of an experienced researcher, it is not unusual to obtain measured cell viabilities of 50% or less.
Pancreatic stem cells are cells that are believed to give rise to all of the different endocrine tissues within the pancreas. It is anticipated that research efforts that are presently underway using these cells will eventually lead to cell therapy aimed at eliminating Type I diabetes, a currently incurable disease afflicting millions of individuals. At present, due to the prevalence of this disease, and the associated economic impact, there is an extensive amount of research being conducted in an effort to expand this stem cell population. Pancreatic stem cells are obtained from whole pancreatic tissue through a series of fractionations. The fraction containing the stem cells is isolated from the other fractions and placed into a serum free medium. Currently, there are no methods available to expand these cells in vitro. Rather, the medium simply serves to maintain the cells in culture, and delay cell death. The cells in this fraction, including the stem cells are present as large aggregates of primary tissue. Large aggregates are undesirable since cells rapidly begin to die due to nutrient and oxygen limitations. Thus, in order to ensure that the cells survive, and to isolate the stem cells from the rest of the cells, it is necessary to dissociate the tissue into a single cell suspension. At present, there are no reliable or reproducible methods to accomplish this. Until now, the best method utilized by researchers, and the current accepted practice in this field has been to mechanically dissociate the aggregates. However, this method does not result in the generation of a single cell suspension. Rather, many cell aggregates remain. Significantly increasing the intensity and duration of the mechanical dissociation process does not remove these aggregates, but rather, results in the death of large numbers of otherwise viable cells. Thus, despite being the most commonly used procedure in this field, mechanical dissociation is not ideal.
It has recently been hypothesized that mutations to cells within the relatively quiescent stem cell compartment of mammary tissue results in the generation of breast cancer when rapid mitotic activity ensues. Thus, there has been a significant increase in research activity related to these cells. One difficulty in conducting research with these cells is that they tend to aggregate when placed into serum-free culture. These aggregates (referred to as mammospheres) are comprised of tightly arranged cells which are very difficult to mechanically dissociate into a single cell suspension.
Chinese hamster ovary cells are very well characterized, and are used extensively in many commercial applications. These cells can be induced to grow as both suspended aggregates, or as a monolayer culture in which the cells are attached to a substrate. In both cases there are issues related to generating a single cell suspension. If the cells are attached as a monolayer, then mechanical dissociation is not effective, and enzymatic means (such as trypsin with EDTA) are routinely used to detach the cells, and subsequently break them into single cells. However, enzymatic approaches are known to cause cell damage, or even death.
Embryonic stem cells (ES) are primitive, undifferentiated cells derived from the inner cell mass of a blastocyst. These cells are termed pluripotent as they have the capacity to differentiate and give rise to the multitude of different cell types which comprise an organism. It has been shown that these cells require attachment to a substrate in order to remain undifferentiated in vitro. If not allowed to attach, the cells form aggregates in suspension called embryoid bodies, and start to differentiate within these aggregates. The current state-of-the-art in this field with respect to detaching the cells from the surface of a flask is to use enzymes such as trypsin. However, as described earlier, enzymes can be harmful to the cells.
In view of the aforementioned deficiencies attendant with prior art methods of dissociating cells in both anchorage dependent and anchorage independent cultures, a need exists for a new approach that reduces the negative consequences of mechanical dissociation and enzymatic dissociation.